Because of their inherent insolubility in aqueous phase, lipids are typically found complex to proteins (i.e. serum albumin, apolipoprotein, and intracellular lipid binding proteins, (LBP's). The interactions which occur between proteins and lipids have medical significance in coronary heart disease, viral infection, cancer, and metabolic diseases (e.g. cardiomyopathy). We have constructed a system which will allow us to describe at high resolution (1.0-1.4 A) the atomic details of protein/lipid/solvent interactions and how they relate to ligand affinity and specifity. We will examine the structures of a number of protein:ligand complexes. Site directed mutagenesis will be done to test theories concerning lipid binding. These results will help to understand the biological function of these proteins and the cellular trafficking of lipid. These studies will also provide us the opportunity to investigate a protein structure with a number of different ligands at a level of accuracy never seen before. The proposed research will use techniques in x-ray crystallography, molecular biology, 13C NMR, and EPR for this analysis. These studies will focus on two LBPs which are expressed in the small intestinal lining cells (enterocytes) of rats. These small (15 kDa) proteins are similar in primary structure however they display unique patterns of tissue expression and lipid specificity. They have been named intestinal fatty acid binding protein (I-FABP) and ileal lipid binding protein (I-LBP) and are members of a family of LBPs containing ten members. We have overexpressed, purified to homogeneity, characterized, and crystallized several members of this family. Rat I-FABP is the best characterized of the LBPs. It binds long chain fatty acids (Kd's 1-3 muM) while I-LBP binds both long chain fatty acids and bile acids (Chenodeoxycholic acid). The high resolution tertiary structure of apo- and plamiyate-bound I-FABP have been refined to 2.0 A resolution. The protein consists of 10 anti-parallel Beta-strands composed of two nearly orthogonal Beta-sheets. Thebound fatty acid is between the Beta-sheets completely encased by protein. The five year goal of these studies is to (1) improve the resolution of apo-I-FABP to greater than 1.3 A, (2) refine the tertiary conformation of I-FABP with several ligands, (3) determine the structure of I-FABP by neutron diffractions, and (4) determine the high resolution tertiary structure of I-LBP.